Three-dimensional polymer networks and their use

ABSTRACT

The disclosure provides three-dimensional cross-linked polymer networks transport channels, arrays comprising the networks, processes for making the networks, and uses of the networks and arrays.

1. CROSS-REFERENCE TO RELATED APPLICATIONS

This application claims priority under 35 U.S.C. § 119 to Europeanapplication no. 17176572.0, filed Jun. 19, 2017, the contents of whichare incorporated herein in their entireties by reference thereto.

2. BACKGROUND

U.S. Publication No. 2008/0293592 describes a method for covalentlyimmobilizing probe-biomolecules on organic surfaces by means ofphotoreactive cross-linking agents. The method has in practice proven tobe advantageous particularly because it permits an immobilization ofprobe biomolecules on unreactive surfaces, such as silanized glasssupports and substrates made of standard commercial plastics. A polymeris used in the method described in US 2008/0293592 to form a type ofthree-dimensional network onto which the probe biomolecules can bebonded, either at the network's surface or in the inside of the network.Compared to an organic surface on which the probe biomolecules are onlyimmobilized in two-dimensional form, the three-dimensionalimmobilization of the biomolecules in the polymer and/or copolymernetwork permits a higher density of the probe biomolecules on theorganic surface. This increases the amount of analyte which can bebonded per surface unit of the organic surface. Use of the surface asbiological sensor thus gives rise to a higher measurement accuracy and ahigh measurement dynamic.

However, a disadvantage of the methods and polymer networks described inU.S. 2008/0293592 is that analyte molecules or analyte components whichbind to probe biomolecules arranged on or close to the surface of thepolymer network can block the network. Further analyte molecules oranalyte constituents can then no longer bind as well to as yet unboundprobe biomolecules which are arranged at a greater distance from thesurface of the network in its interior.

Thus, there is a need for improved polymer networks.

3. SUMMARY

This disclosure provides three-dimensional polymer networks comprisingcross-linked polymer chains, e.g., water-soluble polymer chains, and oneor more transport channels. The transport channels permit molecules insolution, e.g., analyte molecules, to access the polymer chains withinthe network. In certain aspects, the polymer chains are cross-linked toprobe molecules, and the transport channels provide a greater surfacearea for binding of analytes to probe molecules.

The networks are suitably covalently attached to a surface. As impliedin the preceding paragraph, one or more probes, such as a biomolecule,can be immobilized on the surface of the network and throughout theinterior of the network, providing a sensor for detecting the presenceof and/or measuring the amount of an analyte in a sample. For example,nucleic acid probes can be used to detect complementary nucleic acidspresent in a sample and antibody probes can be used to detect antigenspresent in a sample. The networks of the disclosure allow for fasterhybridization of a given amount of analyte than networks lackingtransport channels because the transport channels can effectivelyincrease the surface area of the network, exposing more probes to thesample in a given amount of time. Additionally, the networks of thedisclosure can bind more analyte than the same volume of a transportchannel-free network because the transport channels decrease oreliminate the problem whereby analyte or other components of a samplebound to probes at or near the surface of the network block access toprobes located in the interior of the network. Another advantage of thenetworks of the disclosure is that the high amount of analyte loadingmade possible by the transport channels allows for a more sensitivedetection of analyte than may be possible with a transport channel-freenetwork, i.e., the signal to noise ratio can be improved compared totransport channel-free networks because a given amount of analyte can beconcentrated in a smaller network volume. Yet another advantage of thenetworks of the disclosure is that the high analyte loading madepossible by the transport channels allows for quantification of a widerrange of analyte concentrations compared to transport channel-freenetworks.

This disclosure also provides arrays comprising a plurality of thethree-dimensional networks of the disclosure and a substrate. Arrays ofthe disclosure can be used to detect and/or measure one or more analytesin one or more samples simultaneously. The arrays of the disclosure canbe washed and reused, providing a significant cost advantage over singleuse arrays. Another advantage of the arrays of the disclosure is thatthey can be manufactured in a simple manner because all of thecomponents needed to make an individual network can be applied as asingle mixture onto a surface of the substrate during the manufacturingprocess.

This disclosure also provides processes for making the three-dimensionalnetworks and arrays of the disclosure. The three-dimensional networks ofthe disclosure can be made by cross-linking a polymer in the presence ofat least two types of salt crystals, preferably needle-shaped saltcrystals and compact salt crystals, and subsequently dissolving the saltcrystals to leave behind transport channels in the cross-linked polymernetwork. Without being bound by theory, the inventors believe that thepresence of compact salt crystals during cross-linking results in asponge-like polymer with short channels that are penetrated by longchannels created by the presence of the needle-shaped salt crystalsduring cross-linking.

This disclosure also provides processes for using the three-dimensionalnetworks and arrays of the disclosure to detect and/or measure ananalyte in a sample, preferably a liquid sample.

4. BRIEF DESCRIPTION OF THE FIGURES

FIG. 1 shows a diagrammatic representation of a mixture which has aprobe biomolecule (1) and a polymer (3) comprising two photoreactivegroups (4) per molecule dissolved in an aqueous salt solution (asdescribed in Section 5.3.1).

FIG. 2 shows shows a cross-section through a drop of a mixture (5), suchas that shown in FIG. 1, having a surface (10) located at a spot (7) ofa surface (2) which can be situated on a holder (6). The surface ispreferably that of an organic substrate or a substrate with an organiccontaining. The substrate is preferably rigid. The holder can be aheated holder or a chilled holder to permit controlled crystallizationof the salts in the aqueous salt solution.

FIG. 3 shows shows a cross-section through the arrangement shown in FIG.2 after the mixture has been heated and both (a) needle-shaped saltcrystals (8) extending from crystallization germs (9) and (b) compactcrystals (14) have been formed in the salt solution.

FIG. 4 shows shows a cross-section through the arrangement shown in FIG.3 after the mixture has been dried and irradiated with optical radiation(11) to form a polymer network (15) having a surface (16).

FIG. 5 shows shows a diagrammatic representation of the mixture of FIG.1 following irradiation with optical radiation.

FIG. 6 shows shows a cross-section through the arrangement shown in FIG.4 after dissolving the salt crystals in a solvent (12), formingtransport channels in the form of long channels (13) and short channels(19).

FIG. 7 shows a reaction pathway for the formation of p(Dimethyacryamideco Methacryloyl-Benzophenone co Sodium 4-vinylbenzenesulfonate).

FIG. 8 shows a perspective view of a biochip (17) on which polymernetworks (15) are located at spots (7) arranged as a matrix of rows andcolumns. The chip preferably has an organic surface.

FIG. 9 shows a top view of a biochip (17) as shown in FIG. 8, where eachpolymer network (15) has a diameter (D), and where the rows and columnsare separated by a distance Y and a distance X, respectively, measuredfrom the center points of the polymer networks (15).

FIG. 10 shows a biosensor (17′) comprising a flexible substrate band(18) on which polymer networks (15) having a diameter (D) are located atspots (7) separated by distance X measured from the center points of thepolymer networks.

FIGS. 11A-11C show a biochip with 6 rows (A-F) and 6 columns (1-6) ofpolymer networks prior to (FIG. 11A) and after (FIG. 11B) drying, withshows the salt concentrations used to make the polymer networks of rowsD and E show in FIG. 11C. The polymer networks of rows D and E were madeusing an aqueous salt solution containing both sodium phosphate andpotassium phosphate. The remaining rows were made using an aqueous saltsolution containing only sodium phosphate, at a concentration of 350 mM.The polymer networks of rows D and E look more round and homogeneous.

FIGS. 12A-12C show the results of hybridization of a PCR reactionproduct using S. aureus and E. coli-specific primer pairs to arraysaccording to FIG. 12A-FIG. 12C. FIG. 12A shows hybridization to an arrayof PCR product amplified from 100 copies of S. aureus DNA. FIG. 12Bshows hybridization to an array of PCR product amplified from 100 copiesof E. coli DNA. FIG. 12C shows the probe map for the arrays shown inFIG. 12A and FIG. 12B. E. coli=E. coli probe; Salmonella=Salmonellaprobe (to which the E. coli PCR product shows some cross-reactivity);Allstaph=a pan Staphylococcus probe; S. aureus=S. aureus probe; PCRcontrol=probe to detect internal control for PCR amplification process;LL=landing lights, which are fluorophore-labeled oligonucleotidescross-linked to the polymer chains in the networks used as arraycontrols.

FIG. 13 shows quantification of fluorescence signals from hybridizationof PCR product amplified using S. aureus or E. coli-specific primerpairs in the absence of template, representing background “noise”. No. 1represents spot D1, E1; no. 2 represents the spot D1, E2; no. 3represents spot D1, E3; no. 4 represents the spot D1, E4; no. 5represents spot D1, E5; and no. 6 represents the spot D1, E6.

5. DETAILED DESCRIPTION 5.1. Three-Dimensional Polymer Networks

The three-dimensional networks of the disclosure comprise a cross-linkedpolymer, e.g., a polymer according to Rendl et al., 2011, Langmuir27:6116-6123 or US 2008/0293592, the contents of which are incorporatedby reference in their entireties herein. The three-dimensional networksof the disclosure further comprise one or more transport channels andcan optionally further comprise one or more probes immobilized on thenetwork, e.g., by cross-linking to the polymer chains.

The networks of the disclosure can have a mesh size (measured in thehydrated state of the network) of, for example, 5 to 75 nm (e.g., 10 to20 nm, 10 to 30 nm, 10 to 40 nm, 10 to 50 nm, 20 to 30 nm, 20 to 40 nm,20 to 50 nm, 30 to 40 nm, 30 to 50 nm, or 40 to 50 nm). The “hydratedstate of the network” means that the network is at equilibrium withrespect to water absorption, i.e., it absorbs in aqueous solution asmuch water as it emits.

Polymers that can be used to make the networks are described in Section5.1.1. Cross-linkers than can be used to make the networks are describedin Section 5.1.2. Features of the one or more transport channels aredescribed in Section 5.1.3. Probes that can be immobilized on thenetworks are described in Section 5.1.4.

5.1.1. Polymers

The three-dimensional networks of the disclosure can comprise across-linked homopolymer, copolymer, mixtures of homopolymers, mixturesof copolymers, or mixtures of one or more homopolymers and one or morecopolymers. The term “polymer” as used herein includes both homopolymersand/or copolymers. The term “copolymer” as used herein includes polymerspolymerized from two or more types of monomers (e.g., bipolymers,terpolymers, quaterpolymers, etc.). Copolymers include alternatingcopolymers, periodic copolymers, statistical copolymers, randomcopolymers, block copolymers, linear copolymers and branched copolymers.The three-dimensional networks of the disclosure can comprise anycombination of the foregoing types of polymers. Reagents and methods formaking such polymers are known in the art (see, e.g., Ravve, A.,Principles of Polymer Chemistry, Springer Science+Business Media, 1995;Cowie, J. M. G., Polymers: Chemistry & Physics of Modern Materials,2^(nd) Edition, Chapman & Hall, 1991; Chanda, M., Introduction toPolymer Science and Chemistry: A Problem-Solving Approach, 2^(nd)Edition, CRC Press, 2013; Nicholson, J. W., The Chemistry of Polymers,4^(th) Edition, RSC Publishing, 2012; the contents of each of which areherein incorporated by reference in their entirety).

Preferred polymers are hydrophilic and/or contain hydrophilic groups.The polymer is preferably water-soluble. In an embodiment, the polymeris a copolymer that has been polymerized from two or more species ofmonomers selected to provide a desired level of water solubility. Forexample, water solubility of a copolymer can be controlled by varyingthe amount of a charged monomer, e.g., sodium 4-vinylsulfonate, used tomake the copolymer.

When cross-linked, water-soluble polymers form water-swellable gels orhydrogels. Hydrogels absorb aqueous solutions through hydrogen bondingwith water molecules. The total absorbency and swelling capacity of ahydrogel can be controlled by the type and degree of cross-linkers usedto make the gel. Low cross-link density polymers generally have a higherabsorbent capacity and swell to a larger degree than high cross-linkdensity polymers, but the gel strength of high cross-link densitypolymers is firmer and can maintain network shape even under modestpressure.

A hydrogel's ability to absorb water is a factor of the ionicconcentration of the aqueous solution. In certain embodiments, ahydrogel of the disclosure can absorb up to 50 times its weight (from 5to 50 times its own volume) in deionized, distilled water and up to 30times its weight (from 4 to 30 times its own volume) in saline. Thereduced absorbency in saline is due to the presence of valence cations,which impede the polymer's ability to bond with the water molecule.

The three-dimensional network of the disclosure can comprise a copolymerthat has been polymerized from one, two, three, or more than threespecies of monomers, wherein one, two, three or more than three of thespecies of monomers have a polymerizable group independently selectedfrom an acrylate group (e.g., acrylate, methacrylate, methylmethacrylate, hydroxyethyl methacrylate, ethyl acrylate, 2-phenylacrylate), an acrylamide group (e.g., acrylamide, methacrylamide,dimethylacrylamide, ethylacrylamide), an itaconate group (e.g.,itaconate, 4-methyl itaconate, dimethyl itaconate) and a styrene group(e.g. styrene, 4-methyl styrene, 4-ethoxystyrene). Preferredpolymerizable groups are acrylate, methacrylate, ethacrylate, 2-phenylacrylate, acrylamide, methacrylamide, itaconate, and styrene. In someembodiments, one of the monomers used to make the copolymer is charged,e.g., sodium 4-vinylbenzenesulfonate.

The polymer used to make a network of the disclosure can comprise atleast one, at least two, or more than two cross-linker groups permolecule. A cross-linker group is a group that covalently bonds thepolymer molecules of the network to each other and, optionally, toprobes and/or a substrate. Copolymers that have been polymerized fromtwo or more monomers (e.g., monomers having a polymerizable groupindependently selected from those described in the preceding paragraph),at least one of which comprises a cross-linker, are suitable for makinga three-dimensional network of the disclosure. Exemplary cross-linkersare described in Section 5.1.2. A preferred monomer comprising across-linker is methacryloyloxybenzophenone (MABP) (see FIG. 7).

In a preferred embodiment, the copolymer is a bipolymer or a terpolymercomprising a cross-linker. In a particularly preferred embodiment, thecopolymer comprises p(Dimethyacryamide co Methacryloyl-Benzophenone coSodium 4-vinylbenzenesulfonate) (see FIG. 7).

5.1.2. Cross-Linkers

Cross-linking reagents (or cross-linkers) suitable for making thecross-links in the three-dimensional networks include those activated byultraviolet light (e.g., long wave UV light), visible light, and heat.Exemplary cross-linkers activated by UV light include benzophenone,thioxanthones (e.g., thioxanthen-9-one, 10-methylphenothiazine) andbenzoin ethers (e.g., benzoin methyl ether, benzoin ethyl ether).Exemplary cross-linkers activated by visible light include ethyl eosin,eosin Y, rose bengal, camphorquinone and erythirosin. Exemplarycross-linkers activated by heat include 4,4′ azobis(4-cyanopentanoic)acid, and 2,2-azobis[2-(2-imidazolin-2-yl) propane]dihydrochloride, andbenzoyl peroxide. Other cross-linkers known in the art, e.g., thosewhich are capable of forming radicals or other reactive groups uponbeing irradiated, may also be used.

5.1.3. Transport Channels

The three-dimensional networks of the disclosure contain one or moretransport channels.

Transport channels can allow access to the interior of the network.Although transport channels can have a relatively large cross-section,the network can remain mechanically stable because the mesh size of thenetwork can be significantly smaller than the transport channelcross-section.

The transport channels can form a sort of highway, through whichanalytes can enter quickly in and out of the interior of the network.The transport of the analytes can be effected in the transport channelsby diffusion and/or convection.

Transport channels are formed when a network is formed by cross-linkingpolymer chains in the presence of salt crystals, as described in Section5.3. After salt crystals are washed away, transport channels are leftbehind.

Without being bound by theory, the inventors believe that the methods ofmaking the networks in the disclosure result the formation of at leasttwo types of salt crystals resulting from different metal ion—salt ionpairings. When the salt crystals are washed away, at least two types oftransport channels are left behind, according to the principle of the“lost” form. The transport channels allow analytes to penetrate into theinterior of the network and specifically bind a probe located in theinterior of the network. Additionally, the transport channels allowunbound analytes to exit the interior of the network after washing,reducing the amount of nonspecific signal from analytes “stuck” withinthe network.

One type of transport channel is believed to be a long channel createdfrom needle-shaped salt crystals. As used herein, a “long channel” is anelongated passage in a network that (1) is substantially straight, and(2) in the hydrated state of the network, has a minimum cross-sectionthat is at least 300 nm and a length that is at least three times,preferably five times, and more preferably at least ten times, theminimum cross-section of the passage. For example, the length of thelong channel can be 3 to 15 times, 5 to 10 times, or 10 to 15 times theminimum cross-section of the long channel. A long channel that is“substantially straight” is one which extends from a point of nucleationin one direction without changing direction more than 45 degrees in anydirection, i.e., the X, Y or Z direction. Because long channels arisefrom needle-shaped crystals that form from a common nucleation point,the networks of the disclosure might include groups of (e.g., 5, 10 ormore) long channels that converge at a point located within the networkcorresponding to the original nucleation point of crystallization. Longchannels are typically arranged such that, starting from the surface ofthe network towards the interior, the lateral distance between the longchannels decreases.

In other aspects, one type of transport channel is believed to be ashort channel, for example formed from cubic or rod-shaped crystals. Asused herein, a “short channel” is a passage in a network that (1) issubstantially straight, and (2) in the hydrated state of the network,has a minimum cross-section that is preferably at least 10 times themesh size of the network and a length that is less than three times(e.g., can range from 1 time to 2.75 times, from 1 time to 2.5 times,from 1 time to 2 times, or from 1 time to 1.5 times) the minimumcross-section of the passage. A short channel that is “substantiallystraight” is one which extends from a point of nucleation in onedirection without changing direction more than 45 degrees in anydirection, i.e., the X, Y or Z direction. To maintain network strength,a short channel preferably has a cross-section of no greater than1/20^(th) of the network width or diameter, for example for a networkthat is in the form of a “spot” on an array with a diameter of 200 μm,the cross-section of the short channel is preferably no greater than 10μm, and for a spot on an array with a diameter of 100 μm, thecross-section of the short channel is preferably no greater than 5 μm.In certain aspects, the cross-section of the short channel is about 20nm or greater, about 50 nm or greater, about 100 nm or greater, about250 nm or greater, at least 500 nm or greater, or about 1 μm or greater.The short channels in a network can have approximately (e.g., +/−10% or+/−25%) the same diameter or different diameters. In particularembodiments, the short channels in a network have a diameter rangingbetween any two of the foregoing dimensions, e.g., they can range from100 nm to 10 μm, from 50 nm to 1 μm, from 500 nm to 5 μm, from 250 nm to10 μm, and so on and so forth.

Without being bound by theory, the inventors believe that the shortchannels create a sponge polymer that is penetrated by the longchannels.

5.1.4. Probes

A probe immobilized on the network of the disclosure can be abiomolecule or a molecule that binds a biomolecule, e.g., a partner of aspecifically interacting system of complementary binding partners(receptor/ligand). For example, probes can comprise nucleic acids andtheir derivatives (such as RNA, DNA, locked nucleic acids (LNA), andpeptide nucleic acids (PNA)), proteins, peptides, polypeptides and theirderivatives (such as glucosamine, antibodies, antibody fragments, andenzymes), lipids (e.g., phospholipids, fatty acids such as arachidonicacid, monoglycerides, diglycerides, and triglycerides), carbohydrates,enzyme inhibitors, enzyme substrates, antigens, and epitopes. Probes canalso comprise larger and composite structures such as liposomes,membranes and membrane fragments, cells, cell lysates, cell fragments,spores, and microorganisms.

A specifically interacting system of complementary bonding partners canbe based on, for example, the interaction of a nucleic acid with acomplementary nucleic acid, the interaction of a PNA with a nucleicacid, or the enzyme/substrate, receptor/ligand, lectin/sugar,antibody/antigen, avidin/biotin or streptavidin/biotin interaction.

Nucleic acid probes can be a DNA or an RNA, for example, anoligonucleotide or an aptamer, an LNA, PNA, or a DNA comprising amethacryl group at the 5′ end (5′ Acrydite™). Oligonucleotide probes canbe, for example, 12 to 30, 14 to 30, 14 to 25, 14 to 20, 15 to 30, 15 to25, 15 to 20, 16 to 30, 16 to 25, 16 to 20, 15 to 40, 15 to 45, 15 to50, 15 to 60, 20 to 55, 18 to 60, 20 to 50, 30 to 90, 20 to 100, 20 to60, 40 to 80, 40 to 100, 20 to 120, 20 to 40, 40 to 60, 60 to 80, 80 to100, 100 to 120 or 12 to 150 nucleotides long. In preferred embodiments,the oligonucleotide probe is 15 to 60 nucleotides in length.

When using a nucleic acid probe, all or only a portion of the probe canbe complementary to the target sequence. The portion of the probecomplementary to the target sequence is preferably at least 12nucleotides in length, and more preferably at least 15, at least 18 orat least 20 nucleotides in length. For nucleic acid probes of greaterlength than 40 or 50 nucleotides, the portion of the probe complementaryto the target sequence can be at least 25, at least 30 or at least 35nucleotides in length.

The antibody can be, for example, a polyclonal, monoclonal, or chimericantibody or an antigen binding fragment thereof (i.e., “antigen-bindingportion”) or single chain thereof, fusion proteins comprising anantibody, and any other modified configuration of the immunoglobulinmolecule that comprises an antigen recognition site, including, forexample without limitation, single chain (scFv) and domain antibodies(e.g., human, camelid, or shark domain antibodies), maxibodies,minibodies, intrabodies, diabodies, triabodies, tetrabodies, vNAR andbis-scFv (see e.g., Hollinger and Hudson, 2005, Nature Biotech23:1126-1136). An antibody includes an antibody of any class, such asIgG, IgA, or IgM (or sub-class thereof), and the antibody need not be ofany particular class. Depending on the antibody amino acid sequence ofthe constant domain of its heavy chains, immunoglobulins can be assignedto different classes. There are five major classes of immunoglobulins:IgA, IgD, IgE, IgG, and IgM, and several of these may be further dividedinto subclasses (isotypes), e.g., IgG₁, IgG₂, IgG₃, IgG₄, IgA₁ and IgA₂.“Antibody” also encompasses any of each of the foregoingantibody/immunoglobulin types.

Three-dimensional networks of the disclosure can comprise a singlespecies of probe or more than one species of probe (e.g., 2, 3, 4, or 5or more species). Three-dimensional networks can comprise more than onespecies of probe for the same target (e.g., antibodies binding differentepitopes of the same target) and/or comprise probes that bind multipletargets.

The networks can comprise a labeled (e.g., fluorescently labeled)control probe molecule that can be used, for example, to measure theamount probe present in the network.

The probes can be distributed throughout the network (e.g., on a surfaceand the interior of a network). Preferably, at least one probe is spacedaway from the surface of the network and adjoins at least one transportchannel. A probe so located is then directly accessible for analytemolecules or analyte components through the transport channel. In someembodiments, a majority of the probes are located in the interior of thenetwork.

The one or more probes can be immobilized on the network covalently ornon-covalently. For example, a probe can be cross-linked to thecross-linked polymer or a probe can be non-covalently bound to thenetwork (such as by binding to a molecule covalently bound to thenetwork). In a preferred embodiment, one or more probes are cross-linkedto the cross-linked polymer. In some embodiments, a majority of theprobes are covalently bound in the interior of the network (e.g., suchthat at least a portion of the probes adjoin a transport channel).

Without being bound by theory, the inventors believe that the processesdescribed in Section 5.3 for manufacturing three-dimensional networks inthe presence of salt crystals (particularly phosphate salt crystals) mayresult in a greater concentration of probe molecule at or near theinterface between the polymer and the transport channel due toelectrostatic interactions between the probe molecules (particularlynucleic acid probe molecules) and the salt crystals. Accordingly, insome embodiments of the invention, the disclosure provides networksaccording to the disclosure in which the probe density is greater at theinterface between the polymer and the transport channels than withinregions of the polymer not abutting a transport channel. In variousembodiments, the probe density it at least 10%, at least 20%, at least30%, at least 40%, or at least 50% more dense at the interface betweenthe polymer and the transport channels than within regions of thepolymer not abutting a transport channel.

The density of probe molecule in a network can be verified using thefollowing procedure:

The network is brought into contact with an aqueous liquid at roomtemperature, for example, in a bowl. The liquid contains a plurality ofnanoparticles attached to a moiety that interacts with the probemolecules in the network, for example streptavidin if the probemolecules are biotinylated. The size of the nanoparticles is smallerthan the mesh size of the network and smaller than the minimumcross-section of at least one type of transport channel in the networkto allow the nanoparticles to become distributed throughout the polymer.Suitable nanoparticles are quantum dots 2-5 nanometers in diameter.

An incubation period is selected so that the network in the liquid iscompletely hydrated, i.e., that the network on average takes the sameamount of water as it releases. The incubation period can be, forexample, one hour. The penetration of the nanoparticles in the networkcan be accelerated by setting in motion the network and/or the liquidduring the incubation, for example, by vibrating the network and/orliquid, preferably by means of ultrasonic waves.

After completion of the incubation, the liquid is separated from thenetwork, for example, by draining the liquid from the bowl or taking thenetwork out of the bowl.

Then, the hydrated network is frozen, for example, by means of liquidnitrogen. Thereafter, the frozen network can be cut with the aid of acryomicrotome along mutually parallel cutting planes into thin slices.The cutting planes are arranged transversely to the longitudinalextension of the transport channel and penetrate the transport channel.The cutting is preferably carried out using a liquid nitrogen-cooleddiamond blade. The thickness of the slices can be, for example, about100 nm or 200 nm.

With the aid of a microscope, the nanoparticles disposed in the disksobtained by cutting the frozen network are located. The nanoparticlescan be fluorescent and optically highlighted so that they can be betterdistinguished from the network, if necessary. The locating of thenanoparticles can be done using a suitable software with imageprocessing methods. To examine the disks, preferably a confocalmicroscope laser scanning microscope with fluorescence optics or anelectron microscope is used.

The geometry and/or position information of the nanoparticles obtainedin this manner may be, with the aid of a computer, used to make athree-dimensional geometric model of distribution of the nanoparticlesin the network. The model can then be used to determine whether thedistribution of nanoparticles reflects a greater density of probemolecules near sites of transport channels.

5.2. Arrays

The three-dimensional networks of the disclosure can be positioned(e.g., deposited) on a substrate, and are preferably immobilized on asubstrate (e.g., by covalent cross-links between the network and thesubstrate). A plurality of networks can be immobilized on a substrate toform an array useful, for example, as a biochip.

Suitable substrates include organic polymers, e.g., cycloolefincopolymers (COCs), polystyrene, polyethylene, polypropylene,polycarbonate, and polymethylmethacrylate (PMMA, Plexiglas®). Ticonamarkets an example of a suitable COC under the trade name Topas®.Inorganic materials (e.g., metal, glass) can also be used as asubstrate. Such substrates can be coated with organic molecules to allowfor cross-links between the network and a surface of the substrate. Forexample, inorganic surfaces can be coated with self-assembled monolayers(SAMs). SAMs can themselves be completely unreactive and thus compriseor consist of, for example, pure alkyl silanes. Other substrates canalso be suitable for cross-linking to the three-dimensional networkprovided they are able to enter into stable bonds with organic moleculesduring free-radical processes (e.g., organoboron compounds).

The substrate can be rigid or flexible. In some embodiments, thesubstrate is in the shape of a plate (e.g., a rectangular plate, asquare plate, a circular disk, etc.). For example, the substrate cancomprise a microwell plate, and the three-dimensional networks can bepositioned in the wells of the plate.

The individual networks can be positioned at distinct spots on a surfaceof the substrate, e.g., in a matrix comprising a plurality of columnsand rows. In the embodiment shown in FIG. 8, the networks are located at36 spots arranged in six columns and six rows. Arrays having differentnumbers of rows and columns, the number of each of which can beindependently selected, are contemplated (e.g., 2 to 64 columns and 2 to64 rows). The columns can be separated by a distance X and the rows canbe separated by a distance Y (for example, as shown in FIG. 9) so as toform a grid of spots on which the individual networks can be located. Xand Y can be selected so that the networks, located at the spots of thegrid, do not contact each other in the dehydrated state and do notcontact each other in the hydrated state. The dimensions X and Y can bethe same or different. In some embodiments, X and Y are the same. Insome embodiments, X and Y are different. In some embodiments, X and Yare independently selected from distances of at least about 500 μm(e.g., 500 μm to 5 mm, 500 μm to 4 mm, 500 μm to 3 mm, 500 μm to 2 mm,or 500 μm to 1 mm). In some embodiments, X and Y are both about 500 μm.In other embodiments, X and Y are both 500 μm.

In some embodiments, substrate is band-shaped (for example, as shown inFIG. 10). The networks can be arranged as a single row extending in thelongitudinal direction of a band-shaped organic surface, or can bearranged as multiple rows extending in the longitudinal direction of theband-shaped surface. The rows and columns in such band-shaped arrays canhave grid dimensions X and Y as described above.

The individual networks can each cover an area of the surface of thearray that is circular or substantially circular. Typically, thediameter of the area on the surface of the array covered by theindividual networks (i.e., the spot diameter) is 80 μm to 1000 μm. Invarious embodiments, the spot diameter is 80 μm, 100 μm, 120 μm, 140 μm,160 μm, 180 μm, 200 μm, 300 μm, 400 μm, 500 μm, 600 μm, 700 μm, 800 μm,900 μm, or 1000 μm, or selected from a range bounded by any two of theforegoing embodiments, e.g., 80 μm to 200 μm, 100 μm to 120 μm, 120 μmto 140 μm, 120 μm to 180 μm, 140 μm to 160 μm, 160 μm to 180 μm, 180 μmto 200 μm, 120 μm to 200 μm, 100 μm to 400 μm, 160 μm to 600 μm, or 120μm to 700 μm, and so on and so forth. In a preferred embodiment, thediameter ranges from 100 μm to 200 μm or a subrange thereof.

The arrays of the disclosure typically have at least 8 individualthree-dimensional networks. In certain aspects, the arrays have at least16, at least 24, at least 48, at least 96, at least 128, at least 256,at least 512, or at least 1024 individual three-dimensional networks. Insome embodiments, the arrays of the disclosure have 24, 48, 96, 128,256, 512, 1024, 2048, 4096 or 8192 individual networks, or have a numberof three-dimensional networks selected from a range bounded any two ofthe foregoing embodiments, e.g., from 8 to 128, 8 to 512, 24 to 8192, 24to 4096, 48 to 2048, 96 to 512, 128 to 1024, 24 to 1024, 48 to 512, 96to 1024, or 128 to 512 three-dimensional networks, and so on and soforth. In a preferred embodiment, number of three-dimensional networkson an array ranges from 8 to 1024. In a particularly preferredembodiment, the number of three-dimensional networks on an array rangesfrom 25 to 400.

The individual networks which comprise the arrays of the disclosure canhave identical or different probes (e.g., each network can have a uniqueset of probes, multiple networks can have the same set of probes andother networks can have a different set or sets of probes, or all ofnetworks can have the same set of probes). For example, networksarranged in the same row of a matrix can comprise the same probes andthe networks arranged in different rows of the matrix can have differentprobes.

Typically, the individual networks on an array vary by no more than 20%,no more than 15%, no more than 10% or no more than 5% from one anotherby spot diameter and/or network volume.

In some embodiments, the arrays comprise one or more individual networks(e.g., spots on an array) with one or more control oligonucleotides orprobe molecules. The control oligonucleotides can be labelled, e.g.,fluorescently labelled, for use as a spatial control (for spatiallyorienting the array) and/or a quantifying the amount of probe moleculesbound to the networks, for example, when washing and reusing an array ofthe disclosure (i.e., as a “reusability control”). The spatial andreusability control probes can be the same or different probes.

The same spot on the array or a different spot on the array can furtherinclude an unlabelled probe that is complementary to a known target.When used in a hybridization assay, determining the signal strength ofhybridization of the unlabelled probe to the labelled target candetermine the efficiency of the hybridization reaction. When anindividual network (i.e., a spot on an array) is used both as areusability and/or spatial control and a hybridization control, adifferent fluorescent moiety can be used to label the target moleculethan the fluorescent moiety of the reusability control or spatialcontrol probes.

In some embodiments, the arrays of the disclosure can be reused at least5 times, at least 10 times, at least 20 times, at least 30 times, atleast 40 times, or at least 50 times (e.g., 5 to 20 times, 5 to 30times, 10 to 50 times, 10 to 20 times, 10 to 30 times, 20 to 40 times,or 40 to 50 times, preferably comprising reusing the array 10 to 50times). The array can be washed with a salt solution under denaturatingconditions (e.g., low salt concentration and high temperature). Forexample, the array can be washed with a 1-10 mM phosphate buffer at80-90° C. between uses. The temperature of the wash can be selectedbased upon the length (Tm) of the target:probe hybrid.

The integrity of an array can be determined by a “reusability control”probe. The reusability control probe can be fluorescently labeled or canbe detected by hybridization to a fluorescently labeled complementarynucleic acid. The fluorescent label of a fluorescently labeledreusability control probe may be bleached by repeated excitation, beforethe integrity of the nucleic acid is compromised; in such cases anyfurther reuses can include detection of hybridization to a fluorescentlylabeled complementary nucleic acid as a control. Typically, an array ofthe invention is stable for at least 6 months.

In various embodiments, a fluorescently labeled reusability controlprobe retains at least 99%, 95% 90%, 80%, 70%, 60%, or 50% of itsinitial fluorescence signal strength after 5, 10, 20, 30, 40, or 50uses. Preferably, the reusability control probe retains least 75% of itsfluorescence signal strength after 5 or 10 uses. An array can continueto be reused until the reusability control probe retains at least 50% ofits fluorescence signal strength, for example after 20, 30, 40 or 50reuses. The fluorescent signal strength of the control probe can betested between every reuse, every other reuse, every third reuse, everyfourth reuse, every fifth reuse, every sixth reuse, every seventh reuse,every eighth reuse, every ninth reuse, every tenth reuse, or acombination of the above. For example, the signal strength can be testedperiodically between 5 or 10 reuses initially and the frequency oftesting increased with the number of reuses such that it is tested aftereach reuse after a certain number (e.g., 5, 10, 20, 30, 40 or 50) uses.In some embodiments, the frequency of testing averages once per 1, 1.5,2, 2.5, 3, 4, 5 or 10 uses, or averages within a range bounded betweenany two of the foregoing values, e.g., once per 1-2 uses, once per 1-1.5uses, once per 1-3 uses, or once per 1.5-3 uses.

It is noted that the nomenclature of “spatial control”, “reusabilitycontrol” and “hybridization control” is included for convenience andreference purposes and is not intended to connote a requirement that theprobes referred to “spatial control”, “reusability control” and“hybridization control” be used as such.

5.3. Processes For Making Three-Dimensional Polymer Networks

In one aspect, the processes of the disclosure for makingthree-dimensional polymer networks comprise (a) exposing a mixturecomprising an aqueous salt solution, a polymer, a cross-linker and,optionally, one or more probes to salt crystal forming conditions, (b)exposing the mixture to cross-linking conditions to cross-link thepolymer for form a cross-linked polymer network, and (c) contacting thecross-linked polymer network with a solvent to dissolve the saltcrystals and form one or more transport channels.

The processes can further comprise a step of forming the mixture bycombining an aqueous salt solution, a polymer, a cross-linker and,optionally, one or more probes, and/or further comprise a step ofapplying the mixture to a substrate (e.g., a substrate described inSection 5.2) prior to exposing the mixture to salt crystal formingconditions. If the polymer being used has a pre-attached cross-linker(e.g., when using a copolymer polymerized from a monomer comprising across-linker), the step of forming the mixture can comprise combining anaqueous salt solution with the polymer and, optionally, one or moreprobes.

The mixture can be applied to a substrate prior to exposing the mixtureto salt crystal forming conditions for example, by spraying the mixtureonto a surface of the substrate (e.g., at 1024 sites on the surface).The mixture can be applied to the surface using a DNA chip spotter orinkjet printer, for example. In a preferred embodiment, the mixture issprayed using an inkjet printer. This permits a simple and rapidapplication of the mixture to a large number of spots on the substrate.The spots can be arranged, for example, in the form of a matrix inseveral rows and/or columns. Preferably, the salt content in the mixtureduring printing is below the solubility limit so that the mixture doesnot crystallize in the printing head of the printer. The volume ofmixture applied at individual spots can be, for example, 100 pl, 200 pl,300 pl, 400 pl, 500 pl, 750 pl, 1 nl, 2 nl, 3 nl, 4 nl, or 5 nl, or canbe selected from a range bounded by any two of the foregoing values(e.g., 100 pl to 5 nl, 100 pl to 1 nl, 300 pl to 1 nl, 200 pl to 750 nl,100 pl to 500 pl, 200 pl to 2 nl, 500 pl to 2 nl 1 nl to 2 nl, and so onand so forth). In preferred embodiments, the spot volume is 200 pl to 4nl.

The diameter of the individual spots will depend on the composition ofthe mixture, the volume of the mixture applied, and the surfacechemistry of the substrate. Spot diameters typically range between 80 μmto 1000 μm and can be obtained by varying the foregoing parameters. Invarious embodiments, the spot diameters are 80 μm, 100 μm, 120 μm, 140μm, 160 μm, 180 μm, 200 μm, 300 μm, 400 μm, 500 μm, 600 μm, 700 μm, 800μm, 900 μm, or 1000 μm, or selected from a range bounded by any two ofthe foregoing embodiments, e.g., 80 μm to 200 μm, 100 μm to 120 μm, 120μm to 140 μm, 120 μm to 180 μm, 140 μm to 160 μm, 160 μm to 180 μm, 180μm to 200 μm, 120 μm to 200 μm, 100 μm to 400 μm, 160 μm to 600 μm, or120 μm to 700 μm, and so on and so forth. In a preferred embodiment, thediameter ranges from 100 μm to 200 μm or a subrange thereof.

Suitable polymers, cross-linkers, and probes that can be used in theprocesses of the disclosure are described in Sections 5.1.1, 5.1.2, and5.1.4, respectively. In some embodiments, the polymer used in theprocesses has at least one cross-linker group per polymer molecule. In apreferred embodiment, the polymer has at least two cross-linker groupsper molecule. In a particularly preferred embodiment, the polymer has atleast two photoreactive cross-linker groups per molecule. In theseembodiments, separate polymer and cross-linker molecules are not needed.

Suitable salts that can be included in the mixture are described inSection 5.3.1. Suitable salt crystal forming conditions are described inSection 5.3.2. Suitable cross-linking conditions are described inSection 5.3.3. Suitable solvents for dissolving the salt crystals aredescribed in Section 5.3.4.

5.3.1. Salt

The polymer networks of the disclosure are characterized by transportchannels that result when the polymers are cross-linked in a mixturecontaining salt crystals formed from an aqueous solution containing atleast two types of salts.

The salts are preferably selected for their compatibility with one ormore probes. Ideally, each salt has one or more of the followingcharacteristics, (i) the salt is not toxic to the probes (e.g., the saltdoes not denature the probes), (ii) the salt does not react chemicallywith the probes, (iii) the salt does not attack fluorophores, such ascyanine dyes, which are suitable for the optical marking of probes,and/or (iv) the salt does not react with analytes, detection molecules,and/or binding partners bonded thereto. Preferably, at least one of thesalts forms needle-shaped crystals.

In a preferred embodiment, the aqueous salt solution comprises at leasttwo types of monovalent cations, for example two types of alkali metalcations. Alkali metal cations that can be used include sodium cationsand potassium cations, although other alkali metal cations, such aslithium cations, can also be used.

For optimal signal:noise ratio for detection of nucleic acid analytes,the aqueous salt solution preferably comprises sodium and potassiumcations and/or has a total monovalent cation concentration such thatwhen combined with the polymer solution and optional probe solution(prior to cross-linking) the resulting mixture has a total monovalentcation concentration of at least 500 mM. In particular embodiments, thesodium ion concentration in the mixture is at least 250 mM, and mayrange from 250 mM to 500 mM, more preferably is in the 300 mM to 400 mMrange. In a specific embodiment, the sodium ion concentration in themixture is 350 mM. The potassium ion concentration in the mixture ispreferably at least 150 mM, and preferably is in the range of 150 mM to500 mM, more preferably is in the range of 200 mM to 400 mM, and yetmore preferably is in the range of 250 mM to 350 mM.

The aqueous salt solution can be made using a disodium hydrogenphosphate (Na₂HPO₄) and/or sodium dihydrogen phosphate (NaH₂PO₄) which,in aqueous solution, releases Na⁺ cations and phosphate ions PO₄ ³⁻. Theaqueous salt solution can also be made using dipotassium hydrogenphosphate (K₂HPO₄) and/or potassium dihydrogen phosphate (KH₂PO₄).

Preferably, the aqueous salt solution can be a sodium phosphate buffercontaining both disodium hydrogen phosphate and sodium dihydrogenphosphate, supplemented with dipotassium hydrogen phosphate (K₂HPO₄)and/or potassium dihydrogen phosphate (KH₂PO₄). In one embodiment, asodium phosphate buffer containing both disodium hydrogen phosphate andsodium dihydrogen phosphate and a potassium phosphate buffer containingboth dipotassium hydrogen phosphate and potassium dihydrogen phosphateare made separately and combined into a single aqueous solution, priorto or after mixing with the polymer and/or probe solutions.

Generally, the aqueous salt solution preferably has a pH ranging from 6to 9, and more preferably in the range of 7-8.5. In certain exemplaryembodiments, the pH is 7.5, 8, or 8.5, most preferably 8.

For networks containing protein-based probe biomolecules, the aqueoussalt solution can include phosphate buffered saline (“PBS”) and/or amonovalent cation sulfate.

5.3.2. Salt Crystal Forming Conditions

Salt crystal forming conditions can comprise dehydrating the mixture orcooling the mixture until the relative salt content in the mixtureincreases to above the solubility limit, meaning that the mixture issupersaturated with the salt. This promotes the formation of saltcrystals from a crystallization germ located in the volume of themixture towards the surface of the mixture. It is believed, withoutbeing bound by theory, that the use of aqueous solutions containing atleast two different monovalent metal ions results in the formation of atleast two different types of salt crystals.

The mixture can be dehydrated by heating the mixture, exposing themixture to a vacuum, and/or reducing the humidity of the atmospheresurrounding the mixture.

The mixture can be heated by placing the mixture on a heated substrateor surface (e.g., between about 50° C. to about 70° C.), heating thesubstrate or surface on which the mixture has been placed (e.g., tobetween about 50° C. to about 70° C.), and/or contacting the mixturewith a hot gas (e.g., air, nitrogen, or carbon dioxide having atemperature that is higher than the temperature of the mixture) suchthat water is evaporated from the mixture. The contacting with the hotgas can, for example, take place by placing the mixture in a heatingoven. During the transportation to the heating oven, the mixture can bekept at a humidity of 40% or greater, for example at a relative humidityof approximately 60%, although higher relative humidities, even as highas 75% or greater, are also feasible. Mixtures with higher potassium ionconcentrations can tolerate lower relative humidities, and mixtures withlower potassium salt concentration are preferably kept at higherrelative humidities during transport.

By heating the mixture it is also possible to activate thermallyactivatable cross-linkers, if present, and cross-link the polymerthereby.

In some embodiments, the temperature of the heated substrate and/or airused to dehydrate the mixture is 20° C. or more above the temperature ofthe mixture before heating the mixture, but less than 100° C.

The mixture can be cooled by placing the mixture on a cooled substrateor surface (e.g., between about 5° C. to about 15° C.), cooling thesubstrate or surface on which the mixture has been placed (e.g., tobetween about 5° C. to about 15° C.) and/or bringing it into contactwith a cold gas (e.g., air, nitrogen, or carbon dioxide having atemperature that is lower than the temperature of the mixture). Whencooled, the temperature-dependent solubility limit of the salt in themixture decreases until the mixture is ultimately supersaturated withthe salt. In some embodiments, the mixture is cooled by incubating it ina cold chamber with low humidity (e.g., temperatures between 0° C. and10° C., relative humidity <40%).

The temperature in the mixture is preferably held above the dew point ofthe ambient air surrounding the mixture during the formation of the oneor more salt crystals. This prevents the mixture becoming diluted withwater condensed from the ambient air, which could lead to a decrease inthe relative salt content in the mixture.

5.3.3. Cross-Linking Conditions

The cross-linking conditions can be selected based upon the type ofcross-linker used. For example, when using a cross-linker activated byultraviolet light (e.g., benzophenone, a thioxanthone or a benzoinether), the cross-linking conditions can comprise exposing the mixtureto ultraviolet (UV) light. In some embodiments, UV light having awavelength from about 250 nm to about 360 nm is used (e.g., 260±20 nm or355±20 nm). The use of lower energy/longer wavelength UV light (e.g.,360 nm UV light vs. 254 nm UV light) can require longer exposure times.When using a cross-linker activated by visible light (e.g., ethyl eosin,eosin Y, rose bengal, camphorquinone or erythirosin), the cross-linkingconditions can comprise exposing the mixture to visible light. Whenusing a thermally activated cross-linker (e.g., 4,4′azobis(4-cyanopentanoic) acid, and 2,2-azobis[2-(2-imidazolin-2-yl)propane]dihydrochloride, or benzoyl peroxide), the cross-linkingconditions can comprise exposing the mixture to heat.

The length and intensity of the cross-linking conditions can be selectedto effect cross-linking of polymer molecules to other polymer molecules,cross-linking of polymer molecules to probe molecules (if present), andcross-linking of polymer molecules to substrate molecules or organicmolecules present on the substrate (if present). The length andintensity of cross-linking conditions for a mixture containing probescan be determined experimentally to balance robustness of immobilizationand nativity of probe molecules, for example.

5.3.4. Salt Crystal Dissolution

After cross-linking the polymer, the salt crystals can be dissolved inthe solvent in such a way that at least one transport channel is formedin the network. It is believed, without being bound by theory, that theuse of two types of monovalent salt cations during crystal formationresults in at least two types of crystals, compact crystals and aneedle-shaped crystals. The dissolution of the compact crystals isbelieve to result in short channels that create a sponge-like effect inthe network, pierced by long channels resulting from the dissolution ofthe needle-shaped crystals.

When using an array produced by the method of the disclosure as abiological sensor, a high measurement accuracy and high measurementdynamic are permitted.

The solvent for dissolving the one or more salt crystals can be chosenin such a way that it is compatible to the polymer and probes, ifpresent (e.g., the solvent can be chosen such that it does not dissolvethe polymer and probes). Preferably, the solvent used is a water basedbuffer, such as diluted phosphate buffer. Methanol, ethanol, propanol ora mixture of these liquids can be added to the buffer to facilitate theremoval of unbound polymer from the network.

After the removal of the salt crystals the network can collapse due todrying and can be rehydrated. Drying the network has advantages forshipping and stabilization of probe biomolecules.

5.3.5. Methods of Using the Three-Dimensional Networks

The networks and arrays of the disclosure can be used to determine thepresence or absence of an analyte in a sample, preferably a liquidsample. The disclosure therefore provides methods for determiningwhether an analyte is present in a sample or plurality of samples,comprising contacting a network or array of the disclosure comprisingprobe molecules that are capable of binding to the analyte with thesample or plurality of samples and detecting binding of the analyte tothe probe molecules, thereby determining whether the analyte is presentin the sample or plurality of samples. When arrays comprising differentspecies of probes capable of binding different species of analyte areused in the methods, the presence of the different species of analytescan be determined by detecting the binding of the different species ofanalytes to the probes. In some embodiments, the methods furthercomprise a step of quantifying the amount of analyte or analytes boundto the array.

The analyte can be, for example, a nucleic acid, such as a polymerasechain reaction (PCR) amplicon. In some embodiments, the PCR amplicon isamplified from a biological or environmental sample (e.g., blood, serum,plasma, tissue, cells, saliva, sputum, urine, cerebrospinal fluid,pleural fluid, milk, tears, stool, sweat, semen, whole cells, cellconstituent, cell smear, or an extract or derivative thereof). In someembodiments, the nucleic acid is labeled (e.g., fluorescently labeled).

An analyte placed on the surface of the network can penetrate into theinterior of the network through the transport channel in order tospecifically bind to a probe (e.g., a biomolecule) covalently bondedthere to the polymer. When using the arrays of the disclosure with thenetworks immobilized thereon as biological sensor, a high measurementaccuracy and also a high measurement dynamic is permitted.

The networks and arrays of the disclosure can be regenerated after useas a biosensor and can be used several times (e.g., at 5 times, at least10 times, at least 20 times, at least 30 times, at least 40 times, or atleast 50 times). If the probe molecules are DNA, this can be achieved,for example, by heating the network(s) in an 1× phosphate bufferedsaline to a temperature between 80° C. and 90° C. for about 10 minutes.Then, the phosphate buffered saline can be exchanged for a new phosphatebuffered saline to wash the denatured DNA out of the network(s). If theprobe molecules of the network(s) or array are antigens the network(s)or array can be regenerated by bringing the network(s) into contact with0.1 N NaOH for about 10 minutes. Then, the 0.1 N NaOH can be exchangedfor a phosphate buffered saline to wash the antigens out of the network.Thus, some embodiments of the methods of using the networks and arraysof the disclosure comprise using a network or array that has been washedprior to contact with a sample or a plurality of samples.

5.4. Applications of Arrays of the Disclosure

Because the arrays of the invention achieve economical determination ofthe qualitative and quantitative presence of nucleic acids in a sample,it has immediate application to problems relating to health and diseasein human and non-human animals.

In these applications a preparation containing a target molecule isderived or extracted from biological or environmental sources accordingto protocols known in the art. The target molecules can be derived orextracted from cells and tissues of all taxonomic classes, includingviruses, bacteria and eukaryotes, prokaryotes, protista, plants, fungi,and animals of all phyla and classes. The animals can be vertebrates,mammals, primates, and especially humans. Blood, serum, plasma, tissue,cells, saliva, sputum, urine, cerebrospinal fluid, pleural fluid, milk,tears, stool, sweat, semen, whole cells, cell constituent, and cellsmears are suitable sources of target molecules.

The target molecules are preferably nucleic acids amplified (e.g., byPCR) from any of the foregoing sources).

The arrays of the invention can include probes that are useful to detectpathogens of humans or non-human animals. Such probes includeoligonucleotides complementary at least in part to bacterial, viral orfungal targets, or any combinations of bacterial, viral and fungaltargets.

The arrays of the invention can include probes useful to detect geneexpression in human or non-human animal cells, e.g., gene expressionassociated with a disease or disorder such as cancer, cardiovasculardisease, or metabolic disease for the purpose of diagnosing a subject,monitoring treatment of a subject or prognosis of a subject's outcome.Gene expression information can then track disease progression orregression, and such information can assist in monitoring the success orchanging the course of an initial therapy.

6. EXEMPLARY PROTOCOLS

The following exemplary protocols, which refer to the reference numbersprovided in the figures, are within the scope of the disclosure and canbe used in conjunction with the polymers, cross-linkers and probes ofSections 5.1.1, 5.1.2 and 5.1.4, respectively. Further useful polymers(including co-polymers) and cross-linker groups for use in the followingmethods are described in Rendl et al., 2011, Langmuir 27:6116-6123 andin US 2008/0293592, the contents of which are incorporated by referenceherein. In one embodiment, a polymer mixture according to Section 7.2 isused.

6.1. Exemplary Protocol 1

A plate with a surface (2) that is preferably organic is placed on aholder (6) that is heated. Temperatures between 50° C. and 70° C. aresuitable. A mixture (5) containing a polymer (3), probe biomolecules (1)and an aqueous salt solution is spotted on the organic surface (2) usinga standard DNA chip spotter (e.g., Scienion, Germany). Volumes of 0.5 to4 nl are printed on each spot (7) (see, FIG. 2). The liquid of thesespots dries almost immediately leading to a nucleation of salt crystals(8), (14). After nucleation, needle-shaped salt crystals can extend fromat least one crystallization germ (9) located in the volume of themixture (5) to the surface (10) of the mixture (5) (see, FIG. 3).Additionally, the formation of shorter cubic or rod-shaped crystals (14)is believed to occur (see, FIG. 3). After nucleation of the crystals(8), (14), the spots (7) are irradiated in a UV cross-linker immediatelywith optical UV radiation (11) (see, FIG. 4) such that the probebiomolecules (1) are covalently bonded to the polymer (3), and thepolymer (3) is covalently bonded to the organic surface (2) andcross-linked (see, FIG. 5). Care is taken that the dried, cross-linkedmixture (5) is not attracting moisture to become liquid again.

The dried, cross-linked mixture (5) is then brought into contact with asolvent (12) for the crystals (8) such that at the places at which thecrystals (8), (14) were, long (13) and short (19) channels are formed inthe network (15) comprising the polymer (3) and the probe biomolecules(1) (see, FIG. 6). Thereafter, the solvent (12) is removed. The longchannels (13) can extend from the surface (16) of the network (15) intothe interior of the network (15). The solvent (12) in which the saltcrystals (8), (14) are dissolved is chosen in such a way that it iscompatible to the probe biomolecule (1) and also the polymer (3).Preferably, the solvent (12) used is water based.

6.2. Exemplary Protocol 2

A mixture (5) containing a polymer (3), probe biomolecules (1) and anaqueous salt solution is spotted on an organic surface (2) arranged on aplate using a standard DNA chip spotter (e.g., Scienion, Germany).Volumes of 0.5 to 4 nl are printed on each spot (7) (see, FIG. 2). Theplate with the spots (7) on the surface (2), preferably organic, isplaced on a holder (6) that is chilled (see, FIG. 3). Temperaturesbetween 5° C. and 15° C. are suitable. The liquid of these spots iscooled down to reach an over saturation of the buffer that almostimmediately leads to a nucleation of crystals. After nucleationneedle-shaped salt crystals (8) can extend from at least onecrystallization germ (9) located in the volume of the mixture (5) to thesurface (10) of the mixture (5). Additionally, the formation of shortercubic or rod-shaped crystals (14) is believed to occur (see, FIG. 3).After printing these targets are put in an oven (e.g., at 70° C.) forcomplete drying. After nucleation of the crystals the spots areirradiated in a UV cross-linker immediately with optical UV radiation(11) (see, FIG. 4) such that the probe biomolecules (1) are covalentlybonded to the polymer (3), and the polymer (3) is covalently bonded tothe organic surface (2) and cross-linked. Care is taken that the dried,cross-linked mixture is not attracting moisture to become liquid again.

The dried, cross-linked mixture (5) is then brought into contact with asolvent (12) to dissolve the crystals (8), (14) such that at the placesat which the crystals (8), (14) were, transport channels, e.g., longchannels (13) and short channels (19) are formed in the network (15)comprising the polymer (3) and the probe biomolecules (1). Thereafter,the solvent (12) is removed. The long channels (13) can extend from thesurface (16) of the network (15) into the interior of the network (15).The solvent (12) in which the salt crystals (8), (14) are dissolved ischosen in such a way that it is compatible with the probe biomolecule(1) and the polymer (3). Preferably, the solvent (12) used is waterbased.

As can be seen in FIG. 6 a plurality of long channels (13) and shortchannels (19) can be formed in the network (15). The long channels (13)can extend from the surface (16) of the network (15) to at least onepoint located within the network (15). The long channels (13) can bearranged in such a way that—starting from the surface (16) in thedirection of the interior—the lateral distance between the long channels(13) decreases.

6.3. Exemplary Protocol 3

A mixture (5) containing a polymer (3), probe biomolecules (1) and anaqueous salt solution is printed on a surface (2), preferably organic,of a plate at normal conditions with a humidity ranging from 40-80%,preferably 50-70%. The mixture can contain 350 mM sodium phosphate, pH8, and 250-300 mM potassium phosphate, pH 8, for example. Volumes of 0.5to 4 nl are printed on each spot (7). The moisture content in the printcompartment makes sure the spots (7) stay liquid without crystalformation (i.e., no nucleation takes place). The plate is then put in acontainer, a cardboard box for example. Lids are put on the plate fortransport. The plate with the spots (7) is then put in a drying oven oron a hot plate to rapidly cause nucleation such that needle-shaped saltcrystals (8) extend from at least one crystallization germ (9) locatedin the volume of the mixture toward the surface (10) of the mixture (5).Additionally, the formation of shorter cubic or rod-shaped crystals (14)is believed to occur.

The temperature of the oven/hot plate should be 20° C. or more above theprinting temperature. Temperatures above 100° C. are not necessary.

After drying, the mixture is irradiated to cross-link the polymer (3),probe biomolecules (1), and surface (2).

The dried, cross-linked mixture (5) is then brought into contact with asolvent (12) such that at the places at which the crystals (8), (14)were, long channels (13) and short channels (19) are formed in thenetwork (15) comprising the polymer (3) and the probe biomolecules (1).Thereafter, the solvent (12) is removed. The long channels (13) can areextend from the surface (16) of the network (15) into the interior ofthe network (15). The solvent (12) in which the salt crystals (8), (14)are dissolved is chosen in such a way that it is compatible with theprobe biomolecules (1) and the polymer (3). Preferably, the solvent (12)used is water based.

6.4. Exemplary Protocol 4

Alternatively, a plate with spots (7) on the surface (2), which ispreferably organic, prepared as in exemplary protocol 3 can be cooled toachieve nucleation by putting in a cold chamber with low humidity (e.g.,temperatures <10° C., relative humidity <40%). The drying can beperformed by reducing the humidity or by applying a vacuum afternucleation has started. After nucleation, needle-shaped salt crystals(8) can extend from at least one crystallization germ (9) located in thevolume of the mixture (5) toward the surface (10) of the mixture (5).Additionally, the formation of shorter cubic or rod-shaped crystals (14)is believed to occur. The plate with the spots (7) is put in an oven at60°-70° C. for 1 hour to fully dry the spots. The spots (7) are UVirradiated with 1.00 J @254 nm in a UV cross-linker, i.e. Stratalinker2400. To do this, the plate with the spots (7) can be put into thecenter of the chamber with the shorter side parallel to the door of thechamber. Then, the cover is removed and the cross-linker is started.When machine is finished the array is removed and the cover is replaced.

Alternatively, other UV cross-linkers with the same wavelength (240-270nm, for example) or longer wavelengths, e.g., 360 nm, can be used.

The mixture (5) is then brought into contact with a solvent (12) todissolve the crystals (8), (14) such that at the places at which thecrystals (8), (14) were, long channels (13) and short channels (19) areformed in the network (15) comprising the polymer (3) and the probebiomolecules (1). Thereafter, the solvent (12) is removed. The longchannels (13) can extend from the surface (16) of the network (15) intothe interior of the network (15). The solvent (12) in which the saltcrystals (8), (14) are dissolved is chosen in such a way that it iscompatible with the probe biomolecules (1) the polymer (3). Preferably,the solvent (12) used is water based.

7. EXAMPLES 7.1. Background

Polymer networks made substantially as described herein but containing abuffer of sodium phosphate (“NaPi”) at a concentration of 350 mM withouta second salt can dry in and undergo phase separation if the humidity isnot maintained at 60% or greater during drying on a heating plate. Thisis because at lower humidity levels crystallization can take place in anuncontrolled manner.

It would be desirable to increase the sodium phosphate concentration toavoid uncontrolled crystallization. However, the concentration of NaPicannot be increased significantly as then NaPi crystals couldprecipitate in the printer and block the print nozzle.

Various salts (sodium chloride, sodium bwere used in combination withNaPi test experiments. But none of these gave better signals than theNaPi alone. Usually the hybridization signals of these spots wereinferior (data not shown).

Other attempts to minimize drying in at normal humidity levels entailedtesting phosphate buffered saline, sodium citrate buffer, or potassiumphosphate buffer in lieu of NaPi led to lower hybridization signals inthe polymer networks.

But when NaPi buffer was strengthened by potassium-phosphate buffer (asdescribed in Section 7.2) a stabilization of the liquid spot wasobserved without signal loss, particularly when the potassium phosphateconcentration was greater than 150 mM.

When these experiments were performed there was a surprisingobservation. At higher potassium phosphate levels (150 mM and more) thebackground signal (“noise”) was reduced and at concentrations of 200 mMthe noise almost entirely absent from hybridization reaction (asdescribed in Section 7.3). The reason for this effect is most likelythat the short channels result in a sponge-like polymer matrix that ispierced by long channels from the sodium phosphate. The combination ofthese two structures then improves not only the on-kinetics(hybridization) but also the off-kinetics (washing off unbound or weaklybound material). The lower background signal reduces the backgroundsignal and therefore improves the LOD (limit of detection) of any testperformed using polymer networks made using both sodium phosphate andpotassium phosphate.

7.2. Example 1: Formation of Three-Dimensional Polymer Networks

A 10 mg/ml polymer stock solution was prepared by dissolving 10 mg ofthe cross-linking polymer poly(dimethylacrylamide) co 5%Methacryloyl-Benzophenone co 2.5% Sodium 4-vinylbenzenesulfonate in 1.0ml of DNAse free water. This was achieved by vigorous shaking andvortexing for approximately 5 minutes until all the visible polymer isdissolved. The stock solution was then wrapped in foil to protect itfrom light and placed in a refrigerator overnight to ensure the polymercompletely dissolves and to allow the foam to reduce. The polymer has atleast two photoreactive groups per molecule.

Various mixtures containing 10 mg/ml of the polymer (PDMAA-5% MABP-2.5%SSNa), probe biomolecules (including DNA oligonucleotides with a Cy3fluorescent moiety) and an aqueous salt solution with 350 mM sodiumphosphate buffer and in some cases varying amounts of potassiumphosphate were printed on an organic surface of a plate under 65%humidity. Volumes of 1.6 nl were printed on each spot using ScienionSciflex printer. The plate was then put in a container, a cardboard box.Lids were put on the plate having the organic surface for transport. Theplate with the spots on the organic surface was then put in a dryingoven or on a hot plate (70° C.) to cause nucleation of salt crystals.After drying over a 1-hour period, the plate was irradiated tocross-link the polymer, probe biomolecules, and organic surface.

The plate was washed after printing with 10 mM NaPi Buffer to removeunbound material and then dried and stored. The plate was scanned in aSensovation Fluorescence scanner to visually assess the spot morphology.The resulting images are shown in FIG. 11A (after washing) and FIG. 11B(after drying).

The spots in rows D and E include potassium phosphate in varying amounts(as shown in FIG. 11C), whereas the spots in the remaining rows weregenerated using a salt solution containing only sodium phosphate buffer.The inclusion of potassium phosphate resulted in more homogeneous andround polymer networks than spots made with sodium phosphate only (FIG.11A-FIG. 11B). The inclusion of potassium phosphate also allowscontrolled crystallization when the relative humidity is not increased,for example around normal atmospheric humidity of around 40%.

7.3. Example 2: Hybridization Quality of Polymer Networks

Arrays as described in Example 1 were made using probes for detection ofS. aureus or E. coli.

Primer pairs for amplifying S. aureus and E. coli were used in PCRreactions with 100 copies of S. aureus and E. coli genomic DNA,respectively, as templates, and the PCR products hybridized to arrayscontaining the S. aureus and E. coli probes, respectively. Results areshown in FIG. 12A-12B. FIG. 12A shows hybridization to an array of PCRproduct amplified from 100 copies of S. aureus DNA. FIG. 12B showshybridization to an array of PCR product amplified from 100 copies of E.coli DNA. The probe map for the arrays of FIG. 12A and FIG. 12B is shownin FIG. 12C. This study shows that the signal from polymer networks madeusing an aqueous salt solution containing potassium phosphate as well asthe sodium phosphate buffer have comparable signal to polymer networksmade using an aqueous salt solution containing sodium phosphate only.

Surprisingly, polymer networks made using an aqueous salt solutioncontaining potassium phosphate have a reduced background “noise” ascompared to polymer networks made using an aqueous salt solutioncontaining sodium phosphate only, as shown in FIG. 13. FIG. 13 showsquantification of fluorescence signals from hybridization of PCR productamplified using the same S. aureus or E. coli-specific primer pairs inthe absence of template. Thus any hybridization signal representsbackground “noise”. The background “noise” is absent from polymernetworks made in the presence of the higher concentrations of potassiumphosphate.

8. SPECIFIC EMBODIMENTS

The present disclosure is exemplified by the specific embodiments below.

1. A process for making a three-dimensional hydrogel network,comprising:

-   -   (a) exposing a mixture (optionally positioned on the surface of        a substrate), to salt crystal forming conditions comprising:        -   (i) at least two types of monovalent metal ions having a            total concentration of at least 500 mM,        -   (ii) water-soluble polymer chains,        -   (iii) cross-linker moieties, and        -   (iv) optionally, probe molecules, and thereby forming a            mixture containing one or more salt crystals;    -   (b) exposing the mixture containing one or more salt crystals to        cross-linking conditions, thereby forming a hydrogel containing        one or more salt crystals; and    -   (c) contacting the hydrogel containing one or more salt crystals        with a solvent in which the one or more salt crystals are        soluble, thereby dissolving the salt crystals;

thereby forming the three-dimensional hydrogel network.

2. The process of embodiment 1, wherein the mixture comprises at leasttwo types of monovalent metal ions having a total concentration of 500mM to 1000 mM.

3. The process of embodiment 2, wherein total concentration ofmonovalent metal ions in the mixture is 550 mM to 800 mM.

4. The process of embodiment 3, wherein total concentration ofmonovalent metal ions in the mixture is 600 mM to 750 mM.

5. The process of any one of embodiments 1 to 4, wherein the mixturecomprises two types of monovalent metal ions.

6. The process of embodiment 5, wherein the concentration of eachmonovalent ion is at least 150 mM or at least 200 mM.

7. The process of embodiment 5 or embodiment 6, wherein the monovalentmetal ions are selected from sodium ions, potassium ions, and lithiumions.

8. The process of embodiment 5 or embodiment 6, wherein the monovalentmetal ions are sodium ions and potassium ions.

9. The process of embodiment 8, wherein concentration of sodium ions isat least 300 mM.

10. The process of embodiment 9, wherein the concentration of sodiumions is 300 mM to 500 mM.

11. The process of embodiment 10, wherein the concentration of sodiumions is 300 mM to 400 mM.

12. The process of embodiment 11, wherein the concentration of sodiumions is 350 mM.

13. The process of any one of embodiments 8 to 12, wherein theconcentration of potassium ions is 150 mM to 500 mM.

14. The process of embodiment 13, wherein the concentration of potassiumions is 175 mM to 400 mM.

15. The process of embodiment 14, wherein the concentration of potassiumions is 200 mM to 350 mM.

16. The process of embodiment 15, wherein the concentration of potassiumions is 250 mM to 350 mM.

17. The process of any one of embodiments 1 to 4, wherein the mixturecomprises three types of monovalent metal ions.

18. The process of embodiment 17, wherein the concentration of at leasttwo of the monovalent ions is at least 150 mM each or at least 200 mMeach.

19. The process of embodiment 17 or embodiment 18, wherein themonovalent metal ions are sodium ions, potassium ions, and lithium ions.

20. The process of embodiment 19, wherein the concentration of sodiumions is at least 250 mM.

21. The process of embodiment 20, wherein the concentration of sodiumions is 250 mM to 500 mM.

22. The process of embodiment 21, wherein the concentration of sodiumions is 300 mM to 400 mM.

23. The process of embodiment 22, wherein the concentration of sodiumions is 350 mM.

24. The process of any one of embodiments 19 to 23, wherein theconcentration of potassium ions is 150 mM to 500 mM.

25. The process of embodiment 24, wherein the concentration of potassiumions is 200 mM to 400 mM.

26. The process of embodiment 25, wherein the concentration of potassiumions is 250 mM to 350 mM.

27. The process of any one of embodiments 1 to 26, which furthercomprises, prior to step (a), forming the mixture.

28. The process of embodiment 27, wherein forming the mixture comprisescombining an aqueous salt solution comprising monovalent metal cationsand one or more solutions comprising the water-soluble polymer chains,the cross-linker moieties and, if present, the optional probe molecules.

29. The process of embodiment 28, wherein the water-soluble polymerchains and the cross-linker moieties are in a single solution.

30. The process of embodiment 29, wherein the cross-linked moieties arecovalently attached to the polymer chains.

31. The process of any one of embodiments 28 to 30, wherein the aqueoussalt solution has a pH ranging from 6 to 9.

32. The process of embodiment 31, wherein the aqueous salt solution hasa pH ranging from 7 to 8.5.

33. The process of embodiment 32, wherein the aqueous salt solution hasa pH of 8.

34. The process of any one of embodiments 28 to 33, wherein the aqueoussalt solution comprises a solution produced by a process comprisingdissolving disodium hydrogen phosphate, sodium dihydrogen phosphate,dipotassium hydrogen phosphate, potassium dihydrogen phosphate, sodiumsulfate, potassium sulfate or a combination thereof in water or anaqueous solution.

35. The process of embodiment 34, wherein the aqueous salt solution isproduced by a process comprising dissolving disodium hydrogen phosphate,sodium dihydrogen phosphate, dipotassium hydrogen phosphate, potassiumdihydrogen phosphate, or a combination thereof in water or an aqueoussolution.

36. The process of embodiment 35, wherein the aqueous salt solution isproduced by a process comprising dissolving disodium hydrogen phosphate,sodium dihydrogen phosphate, dipotassium hydrogen phosphate, andpotassium dihydrogen phosphate in water.

37. The process of any one of embodiments 1 to 36, wherein theconcentration of phosphate ions in the mixture is at least 250 mM.

38. The process of embodiment 37, wherein the concentration of phosphateions in the mixture is 250 mM to 1000 mM.

39. The process of embodiment 38, wherein the concentration of phosphateions in the mixture is 550 mM to 800 mM.

40. The process of embodiment 39, wherein the concentration of phosphateions in the mixture is 600 mM to 750 mM.

41. The process of any one of embodiments 1 to 40, wherein the saltcrystal forming conditions result in formation one or more needle-shapedcrystals such that one or more long channels are produced afterdissolution of the salt crystals.

42. The process of any one of embodiments 1 to 41, wherein the saltcrystal forming conditions result in formation of one or more compactcrystals such that one or more short channels are produced afterdissolution of the salt crystals.

43. The process of any one of embodiments 1 to 42, wherein the saltcrystal forming conditions comprise dehydrating the mixture.

44. The process of embodiment 43, which comprises dehydrating themixture by heating the mixture, exposing the mixture to a vacuum,reducing the humidity of the atmosphere surrounding the mixture, or acombination thereof.

45. The process of embodiment 44, which comprises dehydrating themixture by exposing the mixture to a vacuum.

46. The process of embodiment 44, which comprises dehydrating themixture by heating the mixture.

47. The process of embodiment 46, wherein heating the mixture comprisescontacting the mixture with a gas that has a temperature which is higherthan the temperature of the mixture.

48. The process of any one of embodiments 1 to 42, wherein the saltcrystal forming conditions comprise cooling the mixture until themixture becomes supersaturated with the salt.

49. The process of embodiment 48, which comprises cooling the mixture bycontacting the mixture with a gas that has a temperature which is lowerthan the temperature of the mixture.

50. The process of any one of embodiments 1 to 49, wherein thetemperature of the mixture during step (a) is maintained above the dewpoint of the atmosphere surrounding the mixture.

51. The process of any one of embodiments 1 to 50, wherein thecross-linker moieties activated by ultraviolet (UV) light and thecross-linking conditions comprise exposing the mixture to ultravioletlight.

52. The process of any one of embodiments 1 to 50, wherein thecross-linker moieties are activated by visible light and thecross-linking conditions comprise exposing the mixture to visible light.

53. The process of any one of embodiments 1 to 50, wherein thecross-linker moieties are activated by heat and the cross-linkingconditions comprise exposing the mixture to heat.

54. The process of any one of embodiments 1 to 53, wherein thewater-soluble polymer chains comprise homopolymer chains.

55. The process of any one of embodiments 1 to 54, wherein thewater-soluble polymer chains comprise copolymer chains.

56. The process of any one of embodiments 1 to 54, wherein thewater-soluble polymer chains comprise a mixture of homopolymer andcopolymer chains.

57. The process of any one of embodiments 54 to 56, wherein thewater-soluble polymer chains comprise polymer chains polymerized fromone or more species of monomers.

58. The process of embodiment 57, wherein each species of monomercomprises a polymerizable group independently selected from an acrylategroup, a methacrylate group, an ethacrylate group, a 2-phenyl acrylategroup, an acrylamide group, a methacrylamide group, an itaconate group,and a styrene group.

59. The process of embodiment 58, wherein at least one monomer speciesin the water-soluble polymer comprises a methacrylate group.

60. The process of embodiment 59, wherein the at least one monomerspecies comprising a methacrylate group is methacryloyloxybenzophenone(MABP).

61. The process of any one of embodiment 57, wherein the water-solublepolymer comprises a polymer polymerized from dimethylacrylamide (DMAA),methacryloyloxybenzophenone (MABP), and sodium 4-vinylbenzenesulfonate(SSNa).

62. The process of any one of embodiments 1 to 61, wherein thewater-soluble polymer chains are chains of a copolymer comprising thecross-linker moieties.

63. The process of embodiment 62, wherein the water-soluble polymerchains comprise at least two cross-linker moieties per polymer molecule.

64. The process of any one of embodiments 1 to 63, wherein thecross-linker moieties are selected from benzophenone, a thioxanthone, abenzoin ether, ethyl eosin, eosin Y, rose bengal, camphorquinone,erythirosin, 4,4′ azobis(4-cyanopentanoic) acid,2,2-azobis[2-(2-imidazolin-2-yl) propane]dihydrochloride, and benzoylperoxide.

65. The process of embodiment 64, wherein the cross-linker moieties arebenzophenone moieties.

66. The process of any one of embodiments 1 to 65, wherein the solventis water or a water-based buffer.

67. The process of embodiment 66, wherein the solvent is water.

68. The process of embodiment 66, wherein the solvent is a water-basedbuffer.

69. The process of embodiment 68, wherein the water-based buffercomprises phosphate, methanol, ethanol, propanol, or a mixture thereof.

70. The process of any one of embodiments 1 to 69, wherein the mixtureof step (a) further comprises probe molecules.

71. The process of embodiment 70, wherein at least some, the majority orall the probe molecules comprise a nucleic acid, a nucleic acidderivative, a peptide, a polypeptide, a protein, a carbohydrate, alipid, a cell, a ligand, or a combination thereof.

72. The process of embodiment 71, wherein at least some of the probemolecules comprise a nucleic acid or a nucleic acid derivative.

73. The process of embodiment 71, wherein at least a majority of theprobe molecules comprise a nucleic acid or a nucleic acid derivative.

74. The process of embodiment 71, wherein all the probe moleculescomprise a nucleic acid or a nucleic acid derivative.

75. The process of embodiment 70, wherein at least some, the majority orall the probe molecules comprise an antibody, an antibody fragment, anantigen, an epitope, an enzyme, an enzyme substrate, an enzymeinhibitor, a nucleic acid, or a combination thereof.

76. The process of embodiment 75, wherein at least some of the probemolecules comprise a nucleic acid.

77. The process of embodiment 75, wherein at least a majority of theprobe molecules comprise a nucleic acid.

78. The process of embodiment 75, wherein all the probe moleculescomprise a nucleic acid.

79. The process of any one of embodiments 76 to 78, wherein the nucleicacid is an oligonucleotide.

80. The process of embodiment 79, wherein the oligonucleotide is 12 to30 nucleotides long.

81. The process of embodiment 79, wherein the oligonucleotide is 14 to30 nucleotides long.

82. The process of embodiment 79, wherein the oligonucleotide is 14 to25 nucleotides long.

83. The process of embodiment 79, wherein the oligonucleotide is 14 to20 nucleotides long.

84. The process of embodiment 79, wherein the oligonucleotide is 15 to30 nucleotides long.

85. The process of embodiment 79, wherein the oligonucleotide is 15 to25 nucleotides long.

86. The process of embodiment 79, wherein the oligonucleotide is 15 to20 nucleotides long.

87. The process of embodiment 79, wherein the oligonucleotide is 16 to30 nucleotides long.

88. The process of embodiment 79, wherein the oligonucleotide is 16 to25 nucleotides long.

89. The process of embodiment 79, wherein the oligonucleotide is 16 to20 nucleotides long.

90. The process of embodiment 79, wherein the oligonucleotide is 15 to40 nucleotides long.

91. The process of embodiment 79, wherein the oligonucleotide is 15 to45 nucleotides long.

92. The process of embodiment 79, wherein the oligonucleotide is 15 to50 nucleotides long.

93. The process of embodiment 79, wherein the oligonucleotide is 15 to60 nucleotides long.

94. The process of embodiment 79, wherein the oligonucleotide is 20 to55 nucleotides long.

95. The process of embodiment 79, wherein the oligonucleotide is 18 to60 nucleotides long.

96. The process of embodiment 79, wherein the oligonucleotide is 20 to50 nucleotides long.

97. The process of embodiment 79, wherein the oligonucleotide is 30 to90 nucleotides long.

98. The process of embodiment 79, wherein the oligonucleotide is 20 to100 nucleotides long.

99. The process of embodiment 79, wherein the oligonucleotide is 20 to120 nucleotides long.

100. The process of embodiment 79, wherein the oligonucleotide is 20 to40 nucleotides long.

101. The process of embodiment 79, wherein the oligonucleotide is 20 to60 nucleotides long.

102. The process of embodiment 79, wherein the oligonucleotide is 40 to80 nucleotides long.

103. The process of embodiment 79, wherein the oligonucleotide is 40 to100 nucleotides long.

104. The process of embodiment 79, wherein the oligonucleotide is 40 to60 nucleotides long.

105. The process of embodiment 79, wherein the oligonucleotide is 60 to80 nucleotides long.

106. The process of embodiment 79, wherein the oligonucleotide is 80 to100 nucleotides long.

107. The process of embodiment 79, wherein the oligonucleotide is 100 to120 nucleotides long.

108. The process of embodiment 79, wherein the oligonucleotide is 12 to150 nucleotides long.

109. The process of any one of embodiments 1 to 108, further comprising,prior to step (a), a step of applying the mixture to a surface of asubstrate.

110. The process of embodiment 109, wherein the mixture is applied in avolume of in a volume of 100 pl to 5 nl.

111. The process of embodiment 109, wherein the mixture is applied in avolume of in a volume of 100 pl to 1 nl.

112. The process of embodiment 109, wherein the mixture is applied in avolume of in a volume of 200 pl to 1 nl.

113. The process of any one of embodiments 109 to 112, wherein the stepof applying the mixture to the substrate comprises spraying the mixtureonto the surface of the substrate.

114. The process of embodiment 113, wherein the mixture is sprayed by aninkjet printer.

115. The process of any one of embodiments 109 to 114, wherein thesubstrate comprises an organic polymer or an inorganic material having aself-assembled monolayer of organic molecules on the surface.

116. The process of embodiment 115, wherein the substrate comprises anorganic polymer.

117. The process of embodiment 116, wherein the organic polymer isselected from cycloolefin copolymers, polystyrene, polyethylene,polypropylene, polycarbonate, and polymethylmethacrylate.

118. The process of embodiment 117, wherein the substrate comprisespolymethylmethacrylate, polystyrene, or cycloolefin copolymers.

119. The process of embodiment 115, wherein the substrate comprises aninorganic material having an alkyl silane self-assembled monolayer onthe surface.

120. The process of any one of embodiments 109 to 119, wherein thesubstrate comprises a microwell plate.

121. The process of any one of embodiments 109 to 120, wherein thepolymer is cross-linked to the surface in step (b).

122. The process of embodiment 121, in which a water-swellable polymeris produced that is cross-linked to the surface.

123. The process of embodiment 122, wherein the water-swellable polymercan absorb up to 50 times its weight of deionized, distilled water.

124. The process of embodiment 122 or embodiment 123, wherein thewater-swellable polymer can absorb 5 to 50 times its own volume ofdeionized, distilled water.

125. The process of any one of embodiments 122 to 124, wherein thewater-swellable polymer can absorb up to 30 times its weight of saline.

126. The process of any one of embodiments 122 to 125, wherein thewater-swellable polymer can absorb 4 to 30 times its own volume ofsaline.

127. A process for making an array, comprising generating a plurality ofthree-dimensional hydrogel networks by the process of any one ofembodiments 1 to 126 at discrete spots on the surface of the samesubstrate.

128. The process of embodiment 127, wherein the three-dimensionalhydrogel networks are generated simultaneously.

129. The process of embodiment 127, wherein the three-dimensionalhydrogel networks are generated sequentially.

130. The process of any one of embodiments 127 to 129, furthercomprising cross-linking the plurality of three-dimensional hydrogelnetworks to the surface of the substrate.

131. A process for making an array, comprising positioning a pluralityof three-dimensional hydrogel networks produced or obtainable accordingto the process of any one of embodiments 1 to 126 at discrete spots on asurface of the same substrate.

132. The process of any one of embodiments 127 to 131, furthercomprising cross-linking the plurality of three-dimensional hydrogelnetworks to the surface.

133. A process for making an array, comprising positioning a pluralityof three-dimensional hydrogel networks produced or obtainable accordingto the process of any one of embodiments 109 to 126 at discrete spots ona surface of the same substrate.

134. The process of embodiment 133, wherein the positioning comprisesapplying the mixtures from which the three-dimensional hydrogel networksare formed at the discrete spots.

135. The process of any one of embodiments 127 to 134, wherein the spotsare arranged in columns and/or rows.

136. A three-dimensional hydrogel network produced or obtainable by theprocess of any one of embodiments 1 to 126.

137. An array comprising a plurality of three-dimensional hydrogelnetworks according to embodiment 136 on a substrate.

138. An array produced or obtainable by the process of any one ofembodiments 127 to 135.

139. The array of embodiment 137 or embodiment 138 which comprises atleast 8 three-dimensional hydrogel networks.

140. The array of embodiment 137 or embodiment 138 which comprises atleast 16 three-dimensional hydrogel networks.

141. The array of embodiment 137 or embodiment 138 which comprises atleast 24 three-dimensional hydrogel networks.

142. The array of embodiment 137 or embodiment 138 which comprises atleast 48 three-dimensional hydrogel networks.

143. The array of embodiment 137 or embodiment 138 which comprises atleast 96 three-dimensional hydrogel networks.

144. The array of embodiment 137 or embodiment 138 which comprises atleast 128 three-dimensional hydrogel networks.

145. The array of embodiment 137 or embodiment 138 which comprises atleast 256 three-dimensional hydrogel networks.

146. The array of embodiment 137 or embodiment 138 which comprises atleast 512 three-dimensional hydrogel networks.

147. The array of embodiment 137 or embodiment 138 which comprises atleast 1024 three-dimensional hydrogel networks.

148. The array of embodiment 137 or embodiment 138 which comprises 24 to8192 three-dimensional hydrogel networks.

149. The array of embodiment 137 or embodiment 138 which comprises 24 to4096 three-dimensional hydrogel networks.

150. The array of embodiment 137 or embodiment 138 which comprises 24 to2048 three-dimensional hydrogel networks.

151. The array of embodiment 137 or embodiment 138 which comprises 24 to1024 three-dimensional hydrogel networks.

152. The array of embodiment 137 or embodiment 138 which comprises 24three-dimensional hydrogel networks.

153. The array of embodiment 137 or embodiment 138 which comprises 48three-dimensional hydrogel networks.

154. The array of embodiment 137 or embodiment 138 which comprises 96three-dimensional hydrogel networks.

155. The array of embodiment 137 or embodiment 138 which comprises 128three-dimensional hydrogel networks.

156. The array of embodiment 137 or embodiment 138 which comprises 256three-dimensional hydrogel networks.

157. The array of embodiment 137 or embodiment 138 which comprises 512three-dimensional hydrogel networks.

158. The array of embodiment 137 or embodiment 138 which comprises 1024three-dimensional hydrogel networks.

159. The array of any one of embodiments 137 to 158, wherein thethree-dimensional hydrogel networks comprise probe molecules, and two ormore of three-dimensional hydrogel networks comprise different speciesof probe molecules.

160. The array of any one of embodiments 137 to 159, wherein thethree-dimensional hydrogel networks comprise probe molecules, and two ormore three-dimensional hydrogel networks comprise the same species ofprobe molecules.

161. The array of any one of embodiments 137 to 158, wherein thethree-dimensional hydrogel networks comprise probe molecules, and eachof the three-dimensional hydrogel networks comprise the same species ofprobe molecules.

162. The array of any one of embodiments 137 to 161, wherein theplurality of three-dimensional hydrogel networks comprises one or morethree-dimensional hydrogel networks comprising labeled control probemolecules.

163. The array of embodiment 162, wherein the labeled control probemolecules are fluorescently labeled.

164. The array of any one of embodiments 137 to 163, wherein thesubstrate comprises a microwell plate and each well of the microwellplate contains no more than a single three-dimensional hydrogel network.

165. A method for determining whether an analyte is present in a sample,comprising:

-   -   (a) contacting a three-dimensional hydrogel network according to        embodiment 136 or an array of any one of embodiments to 164        comprising probe molecules that are capable of binding to the        analyte with the sample; and    -   (b) detecting binding of the analyte to the probe molecules in        the three-dimensional hydrogel network or array, thereby        determining whether the analyte is present in the sample.

166. The method of embodiment 165, which further comprises washing thenetwork or array comprising probe molecules between steps (a) and (b).

167. The method of embodiment 165 or embodiment 166, which furthercomprises contacting the network or array comprising probe moleculeswith a blocking reagent prior to step (a).

168. The method of any one of embodiments 165 to 167, further comprisingquantifying the amount of analyte bound to the three-dimensionalhydrogel network or array comprising probe molecules.

169. A method for determining whether an analyte is present in eachsample in a plurality of samples, comprising:

-   -   (a) contacting an array of any one of embodiments 137 to 164        comprising probe molecules that are capable of binding to the        analyte with the samples; and    -   (b) detecting binding of the analyte to the probe molecules in        the array, thereby determining whether the analyte is present in        each sample in the plurality of samples.

170. A method for determining whether an analyte is present in eachsample in a plurality of samples, comprising:

-   -   (a) contacting an array of any one of embodiments 137 to 164        comprising probe molecules that are capable of binding to the        analyte with the samples and comprising control probe molecules,        wherein the array has been used and washed prior to step (a);        and    -   (b) detecting binding of the analyte to the probe molecules in        the array, thereby determining whether the analyte is present in        each sample in the plurality of samples.

171. A method for determining whether more than one species of analyteis present in a sample, comprising:

-   -   (a) contacting an array of any one of embodiments 137 to 164        comprising different species of probe molecules that are capable        of binding to the different species of analytes with the sample;        and    -   (b) detecting binding of the analytes to the probe molecules in        the array, thereby determining whether more than one species of        analyte are present in the sample.

172. A method for determining whether more than one species of analyteis present in a sample, comprising:

-   -   (a) contacting an array of any one of embodiments 137 to 164        comprising different species of probe molecules that are capable        of binding to the different species of analytes with the sample        and comprising control probe molecules, wherein the array has        been used and washed prior to step (a); and    -   (b) detecting binding of the analytes to the probe molecules in        the array, thereby determining whether more than one species of        analyte are present in the sample.

173. The method of any one of embodiments 169 to 172, in which:

-   -   (a) the substrate of the array comprises a microwell plate;    -   (b) each well of the microwell plate contains no more than a        single three-dimensional hydrogel network; and    -   (c) contacting the array with the samples comprises contacting        each well with no more than a single sample.

174. The method of any one of embodiments 169 to 173, which furthercomprises washing the array comprising probe molecules between steps (a)and (b).

175. The method of any one of embodiments 169 to 174, which furthercomprises contacting the array comprising probe molecules with ablocking reagent prior to step (a).

176. The method of any one of embodiments 169 to 175, further comprisingquantifying the amount of analyte or analytes bound to the array.

177. The method of any one of embodiments 165 to 176, further comprisingreusing the array.

178. The method of embodiment 177, wherein the array is reused at least5 times.

179. The method of embodiment 177, wherein the array is reused at least10 times.

180. The method of embodiment 177, wherein the array is reused at least20 times.

181. The method of embodiment 177, wherein the array is reused at least30 times.

182. The method of embodiment 177, wherein the array is reused at least40 times.

183. The method of embodiment 177, wherein the array is reused at least50 times.

184. The method of embodiment 178, which comprises reusing the array 5to 20 times.

185. The method of embodiment 178, which comprises reusing the array 5to 30 times.

186. The method of embodiment 178, which comprises reusing the array 10to 50 times.

187. The method of embodiment 178, which comprises reusing the array 10to 20 times.

188. The method of embodiment 178, which comprises reusing the array 10to 30 times.

189. The method of embodiment 178, which comprises reusing the array 20to 40 times.

190. The method of embodiment 178, which comprises reusing the array 40to 50 times.

191. The method of any one of embodiments 177 to 190, which compriseswashing the array between reuses.

192. The method of embodiment 191, wherein the array is washed underdenaturing conditions.

193. The method of embodiment 192 wherein the denaturing conditionscomprise exposing the array to heat.

194. The method of embodiment 192 wherein the denaturing conditionscomprise exposing the array to low salt concentrations.

195. The method of embodiment 192 wherein the denaturing conditionscomprise exposing the array to both heat and low salt concentrations.

196. The method of embodiment 192, wherein the denaturing conditions areremoved prior to reuse.

197. The method of embodiment 196, wherein the denaturing conditionscomprise exposing the array to heat and wherein the temperature islowered prior to reuse.

198. The method of embodiment 196, wherein the denaturing conditionscomprise exposing the array to low salt concentrations and wherein thesalt concentration is increased prior to reuse.

199. The method of embodiment 196, wherein the denaturing conditionscomprise exposing the array to both heat and low salt concentrations andwherein the temperature is lowered and the salt concentration isincreased prior to reuse.

200. The method of any one of embodiments 177 to 199, wherein the arraycomprises at least one three-dimensional hydrogel network comprising afluorescently labelled oligonucleotide as a reusability control.

201. The method of embodiment 200, which comprises testing thefluorescent signal strength.

202. The method of embodiment 201, wherein the reusability controlretains at least 70% of its initial fluorescence signal strength after10 uses.

203. The method of embodiment 202, wherein the reusability controlretains at least 50% of its signal strength after 20 uses.

204. The method of any one of embodiments 200 to 203, wherein the arrayis no longer reused after the reusability control loses more than 50% ofits signal strength.

205. The method of any one of embodiments 165 to 204, wherein analyte isa nucleic acid.

206. The method of embodiment 205, wherein the nucleic acid is apolymerase chain reaction (PCR) amplicon.

207. The method of embodiment 205, wherein the PCR amplicon is amplifiedfrom a biological sample or environmental sample.

208. The method of embodiment 207, wherein the PCR amplicon is amplifiedfrom a biological sample.

209. The method of embodiment 207, wherein the PCR amplicon is amplifiedfrom an environmental sample.

210. The method of embodiment 208, wherein the biological sample is ablood, serum, plasma, tissue, cells, saliva, sputum, urine,cerebrospinal fluid, pleural fluid, milk, tears, stool, sweat, semen,whole cells, cell constituent, cell smear, or an extract or derivativethereof.

211. The method of embodiment 210, wherein the biological sample ismammalian blood, serum or plasma or an extract thereof.

212. The method of embodiment 211, wherein the biological sample ishuman or bovine blood, serum or plasma or an extract thereof.

213. The method of embodiment 210, wherein the biological sample is milkor an extract thereof.

214. The method of embodiment 213, wherein the biological sample iscow's milk or an extract thereof.

215. The method of any one of embodiments 205 to 214, wherein nucleicacid is labeled.

216. The method of embodiment 215, wherein the nucleic acid isfluorescently labeled.

217. A process for making a three-dimensional hydrogel network,comprising:

-   -   (a) exposing a mixture (optionally positioned on the surface of        a substrate), to salt crystal forming conditions comprising:        -   (i) at least two types of monovalent metal ions having a            total concentration of at least 500 mM,        -   (ii) water-soluble polymer chains,        -   (iii) cross-linker moieties, and        -   (iv) optionally, probe molecules, and        -   thereby forming a mixture containing one or more salt            crystals;    -   (b) exposing the mixture containing one or more salt crystals to        cross-linking conditions, thereby forming a hydrogel containing        one or more salt crystals; and    -   (c) contacting the hydrogel containing one or more salt crystals        with a solvent in which the one or more salt crystals are        soluble, thereby dissolving the salt crystals;    -   thereby forming the three-dimensional hydrogel network.

218. The process of embodiment 217, wherein the mixture comprises atleast two types of monovalent metal ions having a total concentration of500 mM to 1000 mM.

219. The process of embodiment 218, wherein the mixture comprises sodiumions at a concentration of 200 mM or greater and potassium ions at aconcentration of 150 mM or greater, optionally wherein:

-   -   (a) the concentration of sodium ions ranges from 300 mM to 400        nM; and    -   (b) the concentration of potassium ions ranges from 200 mM to        350 nM.

220. The process of any one of embodiments 217 to 219, which furthercomprises, prior to step (a), forming the mixture, optionally bycombining an aqueous salt solution comprising monovalent metal cationsand one or more solutions comprising the water-soluble polymer chains,the cross-linker moieties and, if present, the optional probe molecules.

221. The process of embodiment 220, wherein aqueous salt solution has apH ranging from 6 to 9.

222. The process of embodiment 220 or embodiment 221, wherein theaqueous salt solution is produced by a process comprising dissolvingdisodium hydrogen phosphate, sodium dihydrogen phosphate, dipotassiumhydrogen phosphate, and potassium dihydrogen phosphate in water.

223. The process of any one of embodiments 217 to 222, wherein thewater-soluble polymer chains comprise methacrylate groups and at leasttwo cross-linker moieties per molecule, optionally wherein thecross-linker moieties are benzophenone moieties.

224. The process of embodiment 223, wherein the water-soluble polymerchains are polymerized from dimethylacrylamide (DMAA),methacryloyloxybenzophenone (MABP), and sodium 4-vinylbenzenesulfonate(SSNa).

225. The process of any one of embodiments 217 to 224, wherein themixture of step (a) comprises probe molecules, optionally wherein theprobe molecules are nucleic acid molecules.

226. A three-dimensional network obtained or obtainable by the processof any one of embodiments 217 to 225.

227. An array comprising a plurality of three-dimensional networksaccording to embodiment 226, wherein (a) the three-dimensional networksare immobilized on the substrate and (b) each of the three-dimensionalnetworks is located at a separate spot on the substrate, optionallywherein the array can be reused at least 10 times.

228. A process for making an array, comprising generating a plurality ofthree-dimensional hydrogel networks by the process of any one ofembodiments 217 to 225 at discrete spots on the surface of the samesubstrate and cross-linking the networks to the substrate during step(b).

229. A method for determining whether an analyte is present in a sample,comprising:

-   -   (a) contacting a three-dimensional hydrogel network according to        embodiment 226 or an array of embodiment 227, said network or        array comprising probe molecules that are capable of binding to        the analyte with the sample; and    -   (b) detecting binding of the analyte to the probe molecules in        the three-dimensional hydrogel network or array, thereby        determining whether the analyte is present in the sample.

230. The method of embodiment 229, wherein the network or array has beenused and washed at least 10 times prior to step (a) or which furthercomprises reusing the network or array at least 10 times following step(b).

231. The method of embodiment 230, which further comprises quantifyingbinding of the analyte to the probe molecules in the three-dimensionalnetwork.

While various specific embodiments have been illustrated and described,it will be appreciated that various changes can be made withoutdeparting from the spirit and scope of the disclosure(s).

9. CITATION OF REFERENCES

All publications, patents, patent applications and other documents citedin this application are hereby incorporated by reference in theirentireties for all purposes to the same extent as if each individualpublication, patent, patent application or other document wereindividually indicated to be incorporated by reference for all purposes.In the event that there is an inconsistency between the teachings of oneor more of the references incorporated herein and the presentdisclosure, the teachings of the present specification are intended.

What is claimed is:
 1. A process for making a three-dimensional hydrogelnetwork having one or more transport channels, comprising: (a) exposingan aqueous salt-polymer solution to salt crystal forming conditions, theaqueous-polymer salt solution comprising: (i) at least two types ofmonovalent metal ions having a total concentration of at least 500 mM,and (ii) water-soluble polymer chains each comprising at least onecross-linker group, thereby forming a mixture containing one or moresalt crystals; (b) exposing the mixture containing one or more saltcrystals to cross-linking conditions, thereby forming a hydrogelcontaining one or more salt crystals; and (c) contacting the hydrogelcontaining one or more salt crystals with a solvent in which the one ormore salt crystals are soluble, thereby dissolving the salt crystals;thereby forming the three-dimensional hydrogel network having one ormore transport channels.
 2. The process of claim 1, wherein the aqueoussalt-polymer solution is positioned on the surface of a substrate whenexposed to the salt crystal forming conditions.
 3. The process of claim1, wherein the aqueous salt-polymer solution comprises at least twotypes of monovalent metal ions having a total concentration of 500 mM to1000 mM.
 4. The process of claim 3, wherein the aqueous salt-polymersolution comprises sodium ions at a concentration of 200 mM or greaterand potassium ions at a concentration of 150 mM or greater.
 5. Theprocess of claim 4, wherein: (a) the concentration of sodium ions rangesfrom 300 mM to 400 mM; and (b) the concentration of potassium ionsranges from 200 mM to 350 mM.
 6. The process of claim 1, which furthercomprises, prior to step (a), forming the aqueous salt-polymer solutionby combining an aqueous salt solution comprising monovalent metalcations and one or more polymer solutions comprising the water-solublepolymer chains.
 7. The process of claim 6, wherein the aqueous saltsolution has a pH ranging from 6 to
 9. 8. The process of claim 6,wherein the aqueous salt solution is produced by a process comprisingdissolving disodium hydrogen phosphate, sodium dihydrogen phosphate,dipotassium hydrogen phosphate, and potassium dihydrogen phosphate inwater.
 9. The process of claim 1, wherein the water-soluble polymerchains comprise methacrylate groups and at least two cross-linker groupsper molecule.
 10. The process of claim 9, wherein the cross-linkergroups are benzophenone moieties.
 11. The process of claim 9, whereinthe water-soluble polymer chains are polymerized from dimethylacrylamide(DMAA), methacryloyloxybenzophenone (MABP), and sodium4-vinylbenzenesulfonate (SSNa).
 12. The process of claim 1, wherein theaqueous salt-polymer solution of step (a) further comprises probemolecules.
 13. The process of claim 12, wherein the probe molecules arenucleic acid molecules.
 14. The process of claim 13, wherein the aqueoussalt-polymer solution is positioned on the surface of a substrate whenexposed to the salt crystal forming conditions.
 15. The process of claim13, wherein the aqueous salt-polymer solution comprises at least twotypes of monovalent metal ions having a total concentration of 500 mM to1000 mM.
 16. The process of claim 15, wherein the aqueous salt-polymersolution comprises sodium ions at a concentration of 200 mM or greaterand potassium ions at a concentration of 150 mM or greater.
 17. Theprocess of claim 16, wherein: (a) the concentration of sodium ionsranges from 300 mM to 400 mM; and (b) the concentration of potassiumions ranges from 200 mM to 350 mM.
 18. The process of claim 13, whichfurther comprises, prior to step (a), forming the aqueous salt-polymersolution by combining an aqueous salt solution comprising monovalentmetal cations and one or more polymer solutions comprising thewater-soluble polymer chains, and the probe molecules.
 19. The processof claim 18, wherein the aqueous salt solution is produced by a processcomprising dissolving disodium hydrogen phosphate, sodium dihydrogenphosphate, dipotassium hydrogen phosphate, and potassium dihydrogenphosphate in water.
 20. The process of claim 13, wherein thewater-soluble polymer chains comprise methacrylate groups and at leasttwo cross-linker groups per molecule.
 21. The process of claim 20,wherein the cross-linker groups are benzophenone moieties.
 22. Theprocess of claim 20, wherein the water-soluble polymer chains arepolymerized from dimethylacrylamide (DMAA), methacryloyloxybenzophenone(MABP), and sodium 4-vinylbenzenesulfonate (SSNa).
 23. A process formaking an array, comprising generating a plurality of three-dimensionalhydrogel networks by the process of claim 1 at discrete spots on thesurface of the same substrate and cross-linking the networks to thesubstrate during step (b).